Development of genome editing using CRISPR/Cas system in Chlamydomonas reinhardtii and scenedesmus spp.

Name: Kanokphan Panyaporn

LCSH: Kasetsart University -- Theses. M.S. (Genetics) 2017

Classification :.LCCS: QK569.C486

LCSH: Kasetsart University. Department of Genetics -- Theses

LCSH: Chlamydomonas

LCSH: Genomes

LCSH: Genomics

LCSH: Scenedesmaceae

LCSH: CRISPR-associated protein 9

Abstract: Since the discovery of a new breakthrough method for genome editing, the CRISPR/Cas system using Cas9 endonuclease combining with single chimeric RNA known as guide RNA (gRNA) to generate double-stranded breaks and DNA repairs at desire locations, this system has been widely used with high efficiency in various organisms including bacteria, fungi, insects, plants and mammals. However, this system appeared to be problematic and have low efficient in microalgae. In Chlamydomonas, the expression of Cas9 protein is toxic to the algal cell, and genome editing could only be obtained through transient expression or a direct Cas9 ribonucleoprotein transfection. An alternative method for efficient genome editing in microalgae is needed. In this work, the toxicity of Cas9 protein in Chlamydomonas and Scenedesmus sp. was investigated to mitigate this problem. In vitro RNA-Cas9 binding assays showed that Cas9 protein bound to ~100 base RNA and this could potentially be the cause of the cell death. Cell toxicity was confirmed in Chlamydomonas as no transformant was obtained after many rounds of Cas9 transformation. A small number of Scenedesmus transformants were obtained with very low transformation rate, and, after molecular analysis only truncated versions of the transgene were identified indicating the Cas9 toxicity in Scenedesmus. Alternative forms of Cas9 including splitCas9 and CRISPR/Cpf1 were tested by expressing in Scenedesmus sp. Transformants expressing head-split-Cas9 and LbCpf1 and AsCpf1 were obtained. Molecular analysis showed that the transformants contained the full length of the transgenes and potentially expressed the transgenes without cell death. Further analysis is needed to confirm the protein expression their applicability for genome editing in microalgae.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Supachai Vuttipongchaikij

Role: Thesis Advisor

Name: Anongpat Suttangkakul

Role: Thesis Co-Advisor

Created: 2017

Modified: 2023-06-19

Issued: 2023-06-19

วิทยานิพนธ์/Thesis

application/pdf

URL: https://kasetsart.idm.oclc.org/login?url=https://www.lib.ku.ac.th/KUthesis/2560/kanokphan-pan-all.pdf

CallNumber: QK569.C486.K36

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Genetics

Grantor: Kasetsart University

©copyrights Kasetsart University

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	kanokphan-pan-all.pdf	3 MB

ใช้เวลา

0.034426 วินาที